Signalling in malaria parasites. The MALSIG consortium.

Depending on their developmental stage in the life cycle, malaria parasites develop within or outside host cells, and in extremely diverse contexts such as the vertebrate liver and blood circulation, or the insect midgut and hemocoel. Cellular and molecular mechanisms enabling the parasite to sense and respond to the intra- and the extra-cellular environments are therefore key elements for the proliferation and transmission of Plasmodium, and therefore are, from a public health perspective, strategic targets in the fight against this deadly disease. The MALSIG consortium, which was initiated in February 2009, was designed with the primary objective to integrate research ongoing in Europe and India on i) the properties of Plasmodium signalling molecules, and ii) developmental processes occurring at various points of the parasite life cycle. On one hand, functional studies of individual genes and their products in Plasmodium falciparum (and in the technically more manageable rodent model Plasmodium berghei) are providing information on parasite protein kinases and phosphatases, and of the molecules governing cyclic nucleotide metabolism and calcium signalling. On the other hand, cellular and molecular studies are elucidating key steps of parasite development such as merozoite invasion and egress in blood and liver parasite stages, control of DNA replication in asexual and sexual development, membrane dynamics and trafficking, production of gametocytes in the vertebrate host and further parasite development in the mosquito. This article, which synthetically reviews such signalling molecules and cellular processes, aims to provide a glimpse of the global frame in which the activities of the MALSIG consortium will develop over the next three years.

Apical organelles of Apicomplexa: biology and isolation by subcellular fractionation.

The apical organelles are characteristic secretory vesicles of Plasmodium, Toxoplasma, Cryptosporidium and other apicomplexan organisms. They consist of rhoptries, micronemes and dense granules. Recent research has provided much new data concerning their structure, contents, functions and development. All of these organelles contain complex mixtures of proteins, with broad homologies as well as differences in molecular structure between species and genera. Many of the proteins interact with host cell membranes, and are thought to mediate selective adhesion to host cells as well as membrane modification during intracellular invasion. Micronemal proteins are important in the initial selection of host cells, and in enabling gliding motility of the parasites, while rhoptries appear to be more important in parasitophorous vacuole formation. Dense granules are involved predominantly in modifying the host cell after invasion. Research into apical organellar composition and function depends on accurate assignment of molecular identity. This requires the simultaneous application of several complementary approaches including immunolocalisation by light- and electron-microscopy, subcellular fractionation, and transgene expression. The merits and limitations of these different types of approach are discussed, and the importance of cell fractionation methods in characterising apical organelle proteins is stressed.

A conserved subtilisin-like protein TgSUB1 in microneme organelles of Toxoplasma gondii.

Proteolytic processing plays a significant role in the process of invasion by the obligate intracellular parasite Toxoplasma gondii. We have cloned a gene, TgSUB1, encoding for a subtilisin-type serine protease found in T. gondii tachyzoites. TgSUB1 protein is homologous to other Apicomplexan and bacterial subtilisins and is processed within the secretory pathway of the parasite. Initial cleavage occurs in the endoplasmic reticulum, after which the protein is transported to micronemes, vesicles that secrete early during host cell invasion. Upon stimulation of microneme secretion, TgSUB1 is cleaved into smaller products that are secreted from the parasite. This secondary processing is inhibited by brefeldin A and serine protease inhibitors. TgSUB1 is a candidate processing enzyme for several microneme proteins cleaved within the secretory pathway or during invasion.

Proteolytic processing and primary structure of Plasmodium falciparum apical membrane antigen-1.

Plasmodium falciparum apical membrane antigen-1 (PfAMA-1) is a malaria merozoite integral membrane protein that plays an essential but poorly understood role in invasion of host erythrocytes. The PfAMA-1 ectodomain comprises three disulfide-constrained domains, the first of which (domain I) is preceded by an N-terminal prosequence. PfAMA-1 is initially routed to secretory organelles at the apical end of the merozoite, where the 83-kDa precursor (PfAMA-1(83)) is converted to a 66-kDa form (PfAMA-1(66)). At about the time of erythrocyte invasion, PfAMA-1(66) selectively translocates onto the merozoite surface. Here we use direct microsequencing and mass spectrometric peptide mass fingerprinting to characterize in detail the primary structure and proteolytic processing of PfAMA-1. We have determined the site at which processing takes place to convert PfAMA-1(83) to PfAMA-1(66) and have shown that both species possess a completely intact and unmodified transmembrane and cytoplasmic domain. Following relocation to the merozoite surface, PfAMA-1(66) is further proteolytically cleaved at one of two alternative sites, either between domains II and III, or at a membrane-proximal site following domain III. As a result, the bulk of the ectodomain is shed from the parasite surface in the form of two soluble fragments of 44 and 48 kDa. PfAMA-1 is not detectably modified by the addition of N-linked oligosaccharides.

Host cell invasion by malaria parasites.

The complex life cycle of the malaria parasite includes three specialized invasive stages, distinct both in terms of their cellular architecture and in their choice of target host cell. Despite the dissimilarities between these forms, there are clear parallels in the manner by which they enter their respective host cells. Advances in the area of erythrocyte invasion by the malaria merozoite, outlined here by Chetan Chitnis and Mike Blackman and discussed at the Molecular Approaches to Malaria conference, Lorne, Australia, 2-5 February 2000, will undoubtedly impact on our understanding of mechanisms of cell entry by the other invasive forms. Similarly, recent progress in dissecting the functional role of surface proteins expressed by sporozoite and ookinete stages has provided fascinating insights into general aspects of invasion by all invasive stages of apicomplexan parasites.

Immunogenicity and efficacy in aotus monkeys of four recombinant Plasmodium falciparum vaccines in multiple adjuvant formulations based on the 19-kilodalton C terminus of merozoite surface protein 1.

The immunogenicity and protective efficacy of four versions of recombinant C-terminal 19-kDa epidermal growth factor-like region of the major surface protein 1 (rMSP1(19)) of Plasmodium falciparum was studied in Aotus monkeys. Vaccination with each of the four rMSP1(19) constructs elicited high levels of antibodies to MSP1(19) but only one construct, the 19-kDa fragment expressed as a secreted fusion protein from Saccharomyces cerevisiae (yP30P2MSP1(19)), induced a high degree of protective immunity in Aotus nancymai against lethal P. falciparum challenge. Protective formulation required Freund's adjuvant; vaccination with yP30P2MSP1(19) in six other adjuvants that are suitable for human use induced lower levels of antibody response and no protection. These results emphasize the need to continue the search for an adjuvant that is comparable to Freund's adjuvant in potency and is safe for use in humans.

Vaccine efficacy of recombinant Plasmodium falciparum merozoite surface protein 1 in malaria-naive, -exposed, and/or -rechallenged Aotus vociferans monkeys.

Protection against a lethal challenge infection of Plasmodium falciparum was elicited in malaria-naive Aotus vociferans monkeys by vaccination with the C terminus 19-kDa protein of the major merozoite surface protein (MSP-1(19)) fused to tetanus toxoid universal T-cell epitopes P30 and P2. Three of four monkeys were protected against a 10(4)-parasite challenge. Four monkeys were challenged with 10(5) parasites; one self-cured the infection, two were protected against high parasitemia (<2%) but were treated for severe anemia (hematocrit of <25%), and the fourth was not protected. In this model system, anemia appears to be a manifestation of incomplete protection (prolonged low-level parasitemia). Enzyme-linked immunosorbent assay (ELISA) antibody titers correlated with protection. Antibodies from some protected monkeys inhibited secondary processing of MSP-1(42) to MSP-1(33) and MSP-1(19). To mimic the repeated reinfections seen in regions where malaria is endemic, a second malaria parasite challenge was administered 4 months later. All P30P2MSP-1(19)-vaccinated monkeys were protected; thus, a single challenge infection may underestimate vaccine efficacy. ELISA antibody titers correlated with protection against a second infection but had decreased compared to the first challenge. As most target populations for asexual blood-stage malaria vaccines will have been exposed to malaria parasites, a malaria parasite-exposed monkey was vaccinated with P30P2MSP-1(19). This monkey was completely protected, while a malaria parasite-naive P30P2MSP-1(19)-vaccinated monkey self-cured a low-grade parasitemia. Prior malaria parasite infection primed the production of anti-native MSP-1(19) antibodies, which were boosted by vaccination with recombinant P30P2MSP-1(19). Preliminary data suggest that immunogenicity studies of vaccines designed for malaria parasite-exposed populations should also be conducted in malaria parasite-exposed subjects.

Maturation and specificity of Plasmodium falciparum subtilisin-like protease-1, a malaria merozoite subtilisin-like serine protease.

Plasmodium falciparum subtilisin-like protease-1 (PfSUB-1) is a protein belonging to the subtilisin-like superfamily of serine proteases (subtilases). PfSUB-1 undergoes extensive posttranslational proteolytic processing. The primary translation product is converted in the parasite endoplasmic reticulum to p54. This is further processed to p47, which accumulates in secretory organelles within the merozoite. Here, we present a detailed study of this processing. In vitro translated PfSUB-1 showed no capacity to undergo autocatalytic processing. However, parasite extracts contain a protease that cleaves the in vitro translated proprotein between Asp(219) and Asn(220) to form two products of 31 (p31) and 54 kDa; the latter was indistinguishable from authentic p54 and remained complexed with p31 in a noncovalent interaction characteristic of that between a subtilase prodomain and its cognate catalytic domain. Cross-linking studies showed that this complex also exists in the parasite. Expression of PfSUB-1 in recombinant baculovirus also resulted in processing to p54. Mutation of the predicted active site serine abolished processing. Recombinant p54 was secreted in a complex with p31, and could be further converted to p47 in vitro. Conversion required calcium, was an intramolecular autocatalytic process, and involved a second cleavage between Asp(251) and Ala(252). A decapeptide based on sequence flanking Asp(219) was efficiently cleaved by recombinant PfSUB-1. We conclude that PfSUB-1 is a subtilase with an unusual substrate specificity and that it is activated by two autocatalytic processing steps.

Proteases involved in erythrocyte invasion by the malaria parasite: function and potential as chemotherapeutic targets.

Malaria places an increasing burden on global public health resources. In the face of growing resistance of the malaria parasite to available antimalarial drugs, there is a need for new drugs and the identification of new chemotherapeutic targets. The malaria parasite has a complex life cycle which includes a number of obligate intracellular stages. Clinical malaria results from cyclic asexual replication of the blood-stage parasite in circulating erythrocytes of the human host. Erythrocyte entry and host cell rupture require the activity of parasite proteases, and these enzymes are, therefore, attractive targets for rational approaches to new drug development. Malarial proteases play a role in at least two distinct aspects of host cell invasion; modification of parasite proteins involved in host cell recognition and entry; and restructuring of the host cell itself, during and following invasion, and in order to allow parasite release from the host cell. This review details recent advances in the identification of these proteases, describes current understanding of their activation and functional role, and discusses their potential as targets for protease inhibitor-based drugs.

PCR-based gene synthesis as an efficient approach for expression of the A+T-rich malaria genome.

The A+T-rich genome of the human malaria parasite Plasmodium falciparum encodes genes of biological importance that cannot be expressed efficiently in heterologous eukaryotic systems, owing to an extremely biased codon usage and the presence of numerous cryptic polyadenylation sites. In this work we have optimized an assembly polymerase chain reaction (PCR) method for the fast and extremely accurate synthesis of a 2.1 kb Plasmodium falciparum gene (pfsub-1) encoding a subtilisin-like protease. A total of 104 oligonucleotides, designed with the aid of dedicated computer software, were assembled in a single-step PCR. The assembly was then further amplified by PCR to produce a synthetic gene which has been cloned and successfully expressed in both Pichia pastoris and recombinant baculovirus-infected High Five(TM) cells. We believe this strategy to be of special interest as it is simple, accessible and has no limitation with respect to the size of the gene to be synthesized. Used as a systematic approach for the malarial genome or any other A + T-rich organism, the method allows the rapid synthesis of a nucleotide sequence optimized for expression in the system of choice and production of sufficiently large amounts of biological material for complete molecular and structural characterization.

PfSUB-2: a second subtilisin-like protein in Plasmodium falciparum merozoites.

Erythrocyte invasion by the malaria merozoite requires the activity of merozoite proteases. We have previously identified a Plasmodium falciparum protein belonging to the superfamily of subtilisin-like serine proteases, which is expressed in a subset of secretory organelles in free merozoites. Here we describe the identification of a second P. falciparum subtilisin-like merozoite protein. Called PfSUB-2, it is encoded by a single copy gene and is expressed as a large putative type I integral membrane protein which undergoes extensive post-translational processing. The terminal processing product is expressed in an apical location in merozoites. PfSUB-2 may mediate one or more of the serine protease activities known to be associated with erythrocyte invasion.

Merozoite surface protein 1, immune evasion, and vaccines against asexual blood stage malaria.

There is an urgent need for a vaccine against malaria and proteins on the surface of the merozoite are good targets for development as vaccine candidates because they are exposed to antibody. However, it is possible that the parasite has evolved mechanisms to evade a protective immune response to these proteins. Merozoite surface protein 1 (MSP-1) is a candidate for vaccine development and its C-terminal sequence is the target of protective antibody. MSP-1 is cleaved by proteases in two processing steps, the second step releases the bulk of the protein from the surface and goes to completion during successful red blood cell invasion. Antibodies binding to the C-terminus of Plasmodium falciparum MSP-1 can inhibit both the processing and erythrocyte invasion. Other antibodies that bind to either the C-terminal sequence or elsewhere in the molecule are 'blocking' antibodies, which on binding prevent the binding of the inhibitory antibodies. Blocking antibodies are a mechanism of immune evasion, which may be based on antigenic conservation rather than diversity. This mechanism has a number of implications for the study of protective immunity and the development of malaria vaccines, emphasising the need for appropriate functional assays and careful design of the antigen.

A subtilisin-like protein in secretory organelles of Plasmodium falciparum merozoites.

In the vertebrate host, the malaria parasite invades and replicates asexually within circulating erythrocytes. Parasite proteolytic enzymes play an essential but poorly understood role in erythrocyte invasion. We have identified a Plasmodium falciparum gene, denoted pfsub-1, encoding a member of the subtilisin-like serine protease family (subtilases). The pfsub-1 gene is expressed in asexual blood stages of P. falciparum, and the primary gene product (PfSUB-1) undergoes post-translational processing during secretory transport in a manner consistent with its being converted to a mature, enzymatically active form, as documented for other subtilases. In the invasive merozoite, the putative mature protease (p47) is concentrated in dense granules, which are secretory organelles located toward the apical end of the merozoite. At some point following merozoite release and completion of erythrocyte invasion, p47 is secreted from the parasite in a truncated, soluble form. The subcellular location and timing of secretion of p47 suggest that it is likely to play a role in erythrocyte invasion. PfSUB-1 is a new potential target for antimalarial drug development.

Passive immunization with antibodies against three distinct epitopes on Plasmodium yoelii merozoite surface protein 1 suppresses parasitemia.

We have produced monoclonal antibodies against Plasmodium yoelii merozoite surface protein 1 (MSP-1) and have assessed their ability to suppress blood stage parasitemia by passive immunization. Six immunoglobulin G antibodies were characterized in detail: three (B6, D3, and F5) were effective in suppressing a lethal blood stage challenge infection, two (B10 and G3) were partially effective, and one (B4) was ineffective. MSP-1 is the precursor to a complex of polypeptides on the merozoite surface; all of the antibodies bound to this precursor and to an approximately 42-kDa fragment (MSP-142) that is derived from the C terminus of MSP-1. MSP-142 is further cleaved to an N-terminal approximately 33-kDa polypeptide (MSP-133) and a C-terminal approximately 19-kDa polypeptide (MSP-119) comprised of two epidermal growth factor (EGF)-like modules. D3 reacted with MSP-142 but not with either of the constituents MSP-133 and MSP-119, B4 recognized an epitope within the N terminus of MSP-133, and B6, B10, F5, and G3 bound to MSP-119. B10 and G3 bound to epitopes that required both C-terminal EGF-like modules for their formation, whereas B6 and F5 bound to epitopes in the first EGF-like module. These results indicate that at least three distinct epitopes on P. yoelii MSP-1 are recognized by antibodies that suppress parasitemia in vivo.

Antibodies that inhibit malaria merozoite surface protein-1 processing and erythrocyte invasion are blocked by naturally acquired human antibodies.

Merozoite surface protein-1 (MSP-1) of the human malaria parasite Plasmodium falciparum undergoes at least two endoproteolytic cleavage events during merozoite maturation and release, and erythrocyte invasion. We have previously demonstrated that mAbs which inhibit erythrocyte invasion and are specific for epitopes within a membrane-proximal, COOH-terminal domain of MSP-1 (MSP-119) prevent the critical secondary processing step which occurs on the surface of the extracellular merozoite at around the time of erythrocyte invasion. Certain other anti-MSP-119 mAbs, which themselves inhibit neither erythrocyte invasion nor MSP-1 secondary processing, block the processing-inhibitory activity of the first group of antibodies and are termed blocking antibodies. We have now directly quantitated antibody-mediated inhibition of MSP-1 secondary processing and invasion, and the effects on this of blocking antibodies. We show that blocking antibodies function by competing with the binding of processing-inhibitory antibodies to their epitopes on the merozoite. Polyclonal rabbit antibodies specific for certain MSP-1 sequences outside of MSP-119 also act as blocking antibodies. Most significantly, affinity-purified, naturally acquired human antibodies specific for epitopes within the NH2-terminal 83-kD domain of MSP-1 very effectively block the processing-inhibitory activity of the anti-MSP-119 mAb 12.8. The presence of these blocking antibodies also completely abrogates the inhibitory effect of mAb 12.8 on erythrocyte invasion by the parasite in vitro. Blocking antibodies therefore (a) are part of the human response to malarial infection; (b) can be induced by MSP-1 structures unrelated to the MSP-119 target of processing-inhibitory antibodies; and (c) have the potential to abolish protection mediated by anti-MSP-119 antibodies. Our results suggest that an effective MSP-119-based falciparum malaria vaccine should aim to induce an antibody response that prevents MSP-1 processing on the merozoite surface.

Glycosyl-phosphatidylinositols in protozoa structure, biosynthesis and intracellular localisation.

We are investigating the structure and biosynthesis of glycosyl-phosphatidylinositols (GPI) in the protozoa Toxoplasma gondii, Plasmodium falciparum, Plasmodium yoelii and Paramecium primaurelia. This comparison of structural and biosynthesis data should lead us to common and individual features of the GPI-biosynthesis and transport in different organisms.

A 22 kDa protein associated with the Plasmodium falciparum merozoite surface protein-1 complex.

The Plasmodium falciparum merozoite surface protein-1 (MSP-1) is synthesized as a precursor of approximately 195 kDa and is processed to form a complex of polypeptides on the surface of free merozoites. As a result of a second processing event, the entire MSP-1 complex is shed from the surface, apart from a C-terminal fragment that remains anchored to the merozoite membrane. We have identified a 22 kDa protein (p22) on the surface of merozoites by cell surface radioiodination and indirect immunofluorescence assay on unfixed free merozoites. p22 is also a component of the shed MSP-1 complex where it is present in part as a 19 kDa form (p22(19)) as shown by immunochemical and peptide mapping analyses. The soluble complex contains MSP-1-derived polypeptides and p22 in approximately stoichiometrically equal amounts. N-terminal amino acid sequence analyses of p22/p22(19) showed that the protein is not derived from the MSP-1 precursor.

Plasmodium knowlesi: secondary processing of the malaria merozoite surface protein-1.

Secondary processing of the Plasmodium falciparum malaria merozoite surface protein-1 (MSP-1) is defined as a single proteolytic cleavage within the carboxy-terminal membrane-bound component of the MSP-1 protein complex on the free merozoite surface. The N-terminal cleavage product (MSP-1(33)) is shed from the parasite surface along with a number of other polypeptides, whereas the C-terminal processing product remains bound to the merozoite surface and is the only part of MSP-1 detectable in the newly invaded host cell. We report that secondary processing of MSP-1 takes place in a similar manner on invasive merozoites of the simian malaria parasite Plasmodium knowlesi. Processing can take place to a limited extent in pure isolated merozoites; however, within 10 min of the addition of purified invasive merozoites to rhesus erythrocytes, processing and shedding of MSP-1 has gone to completion only in those parasites which have undergone invasion; residual free merozoites remain uniformly reactive with antibodies against MSP-1(33). Successful invasion is therefore associated with complete shedding of MSP-1(33) from the merozoite surface. The nucleotide sequence of the 3' domain of the P. knowlesi MSP-1 gene is also presented.